PROJECT SUMMARY Developing functional engineered blood vessels has been a long-standing goal in tissue engineering and as a bridge to treatment of vascular diseases and disorders. A thorough understanding of the complex multi-step mechanisms governing post-natal vasculogenesis is required for success in this field. Although ground- breaking work ranging from in vitro testing to clinical trials has revealed many important players in this process and uncovered one mechanism for vasculogenesis, another observed phenomenon has yet to be recapitulated in vitro. In general, tissues in need of vascular regeneration are hypoxic. Indeed, hypoxia is a key environmental factor driving production of pro-angiogenic factors. Interestingly, observational findings indicate circulating endothelial progenitor cells (EPCs) are recruited to such hypoxic regions, wherein they attach to the damaged endothelium, egress into the extravascular space, form multicellular clusters, then sprout to anastomose with existing blood vessels and accelerate reperfusion. By incorporating vital microenvironmental factors, such as oxygen gradients and substrate mechanics, into design of an in vitro 3D testing platform, an unprecedented level of biomimicry can be reached and this process can be accurately reproduced. The goal of the proposed work is to develop an in vitro system to study this process and determine the mechanism by which the process occurs, to ultimately design novel therapeutics and advance the functionality of engineered tissues. The aims of the proposed work are as follows: (1) Engineer a hydrogel matrix to study hypoxic cluster- based vasculogenesis; (2) Elucidate mechanisms of hypoxic EPC cluster formation; and (3) Study hypoxic cluster-based vascular network formation through dynamic control of matrix mechanics. These aims bring a multidisciplinary approach to understanding this process, by combining the use of engineering tools with basic science. Upon successful completion of these aims, precise regulation of the cluster-based vasculogenetic process will be possible, thus creating new opportunities in treating vascular diseases and disorders.